The present invention relates generally to gene expression. More particularly, the present invention relates to a method for modulating the production of a protein from a polynucleotide in a CHO cell by replacing at least one codon of the polynucleotide with a synonymous codon that has a higher or lower translation efficiency in the CHO cell than the codon it replaces, or by introducing into the CHO cell a polynucleotide that codes for an iso-tRNA which limits the rate of production of the polypeptide and which corresponds to a codon of the first polynucleotide. Even more particularly, the invention relates to the use of a protein-encoding polynucleotide whose codon composition has been modified for enhanced production of the protein in CHO cells.
The expression of foreign heterologous genes in transformed cells is now commonplace. A large number of mammalian genes, including, for example, murine and human genes, have been successfully expressed in various host cells, including bacterial, yeast, insect, plant and mammalian host cells. Nevertheless, despite the burgeoning knowledge of expression systems and recombinant DNA technology, significant obstacles remain when one attempts to express a foreign or synthetic gene in a selected host cell. For example, translation of a synthetic gene, even when coupled with a strong promoter, often proceeds much more slowly than would be expected. The same is frequently true of exogenous genes that are foreign to the host cell. This lower than expected translation efficiency is often due to the protein coding regions of the gene having a codon usage pattern that does not resemble those of highly expressed genes in the host cell. It is known in this regard that codon utilisation is highly biased and varies considerably in different organisms and that biases in codon usage can alter peptide elongation rates. It is also known that codon usage patterns are related to the relative abundance of tRNA isoacceptors, and that genes encoding proteins of high versus low abundance show differences in their codon preferences.
Codon-optimisation techniques have been developed for improving the translational kinetics of translationally inefficient protein coding regions. Traditionally, these techniques have been based on the replacement of codons that are rarely or infrequently used in the host cell with those that are host-preferred. Codon frequencies can be derived from literature sources for the highly expressed genes of many organisms (see, for example, Nakamura et al., 1996, Nucleic Acids Res 24: 214-215). These frequencies are generally expressed on an ‘organism-wide average basis’ as the percentage of occasions that a synonymous codon is used to encode a corresponding amino acid across a collection of protein-encoding genes of that organism, which are preferably highly expressed.
Typically, codons are classified as: (a) “common” codons (or “preferred” codons) if their frequency of usage is above about 4/3×the frequency of usage that would be expected in the absence of any bias in codon usage; (b) “rare” codons (or “non-preferred” codons) if their frequency of usage is below about ⅔×the frequency of usage that would be expected in the absence of any bias in codon usage; and (c) “intermediate” codons (or “less preferred” codons) if their frequency of usage is in-between the frequency of usage of “common” codons and of “rare” codons. Since an amino acid can be encoded by 2, 3, 4 or 6 codons, the frequency of usage of any selected codon, which would be expected in the absence of any bias in codon usage, will be dependent upon the number of synonymous codons which code for the same amino acid as the selected codon. Accordingly, for a particular amino acid, the frequency thresholds for classifying codons in the “common”, “intermediate” and “rare” categories will be dependent upon the number of synonymous codons for that amino acid. Consequently, for amino acids having 6 choices of synonymous codon, the frequency of codon usage that would be expected in the absence of any bias in codon usage is 16% and thus the “common”, “intermediate” and “rare” codons are defined as those codons that have a frequency of usage above 20%, between 10 and 20% and below 10%, respectively. For amino acids having 4 choices of synonymous codon, the frequency of codon usage that would be expected in the absence of codon usage bias is 25% and thus the “common”, “intermediate” and “rare” codons are defined as those codons that have a frequency of usage above 33%, between 16 and 33% and below 16%, respectively. For isoleucine, which is the only amino acid having 3 choices of synonymous codon, the frequency of codon usage that would be expected in the absence of any bias in codon usage is 33% and thus the “common”, “intermediate” and “rare” codons for isoleucine are defined as those codons that have a frequency of usage above 45%, between 20 and 45% and below 20%, respectively. For amino acids having 2 choices of synonymous codon, the frequency of codon usage that would be expected in the absence of codon usage bias is 50% and thus the “common,” “intermediate” and “rare” codons are defined as those codons that have a frequency of usage above 60%, between 30 and 60% and below 30%, respectively. Thus, the categorisation of codons into the “common,” “intermediate” and “rare” classes (or “preferred,” “less preferred” or “non preferred,” respectively) has been based conventionally on a compilation of codon usage for an organism in general (e.g., ‘human-wide’) or for a class of organisms in general (e.g., ‘mammal-wide’). For example, reference may be made to Seed (see U.S. Pat. Nos. 5,786,464 and 5,795,737) who discloses preferred, less preferred and non-preferred codons for mammalian cells in general. However, the present inventor revealed in WO 99/02694 and in WO 00/42190 that there are substantial differences in the relative abundance of particular isoaccepting transfer RNAs in different cells or tissues of a single multicellular organism (e.g., a mammal or a plant) and that this plays a pivotal role in protein translation from a coding sequence with a given codon usage or composition.
Thus, in contrast to the art-recognised presumption that different cells of a multicellular organism have the same bias in codon usage, it was revealed for the first time that one cell type of a multicellular organism uses codons in a manner distinct from another cell type of the same organism. In other words, it was revealed that different cells of an organism can exhibit different translational efficiencies for the same codon and that it was not possible to predict which codons would be preferred, less preferred or non preferred in a selected cell type. Accordingly, it was proposed that differences in codon translational efficiency between cell types could be exploited, together with codon composition of a gene, to regulate the production of a protein in, or to direct that production to, a chosen cell type. Thus, in order to optimise the expression of a protein-encoding polynucleotide in a particular cell type it is necessary to first determine the translational efficiency for each codon in that cell type, rather than to rely on codon frequencies calculated on an organism-wide average basis, and then to codon modify the polynucleotide based on that determination.